Systematic deletion of adenosine receptors reveals novel roles in inflammation and pyroptosis in THP-1 macrophages.

Macrophages perform the fundamental function of sensing cellular damage, initiating and mediating immune response and tissue repair. Adenine nucleotides are in relatively high abundance in cells and are released from cells during tissue damage that are converted to adenosine in the extracellular environment. The A1, A2A, A2B and A3 adenosine receptors serve to regulate immune function. Despite characterization of the adenosine receptors, a comprehensive examination of adenosine receptor signaling in THP-1 macrophage cells has not been done. Moreover, previous studies employed chemical agonists and antagonists that have the potential for off-target affects. Here we systematically knockdown each of the four known adenosine receptors in THP-1 macrophages using validated siRNA and investigated their function under LPS stimulation. We demonstrate that the A1 receptor is required for adenosine-stimulated IL-10 and IL-1ß secretion indicating an important role of this receptor during resolution of inflammation and tissue repair in these cells. The A1 and A3 receptor were required for IL-6 and IL-1ß secretion showing a net pro-inflammatory role for these receptors. Finally, we present the novel finding that THP-1 macrophages lacking the A2B receptor undergo pyroptosis when exposed to LPS, demonstrating a novel role of the A2B receptor in regulation of programmed cell death during inflammation. This work underscores the fundamental importance of adenosine signaling and provides insight into the independent roles of the adenosine receptors in modulating cytokine signaling.

Targeted and untargeted metabolomics provide insight into the consequences of glycine-N-methyltransferase deficiency including the novel finding of defective immune function.

Fatty liver disease is increasing along with the prevalence of obesity and type-2 diabetes. Hepatic fibrosis is a major health complication for which there are no efficacious treatment options available. A better understanding of the fundamental mechanisms that contribute to the accumulation of fibrosis is needed. Glycine-N-methyltransferase (GNMT) is a critical enzyme in one-carbon metabolism that serves to regulate methylation and remethylation reactions. GNMT knockout (GNMT-/- ) mice display spontaneous hepatic fibrosis and later develop hepatocellular carcinoma. Previous literature supports the idea that hypermethylation as a consequence of GNMT deletion contributes to the hepatic phenotype observed. However, limited metabolomic information is available and the underlying mechanisms that contribute to hepatic fibrogenesis in GNMT-/- mice are still incomplete. Therefore, our goals were to use dietary intervention to determine whether increased lipid load exacerbates steatosis and hepatic fibrosis in this model and to employ both targeted and untargeted metabolomics to further understand the metabolic consequences of GNMT deletion. We find that GNMT mice fed high-fat diet do not accumulate more lipid or fibrosis in the liver and are in fact resistant to weight gain. Metabolomics analysis confirmed that pan-hypermethylation occurs in GNMT mice resulting in a depletion of nicotinamide intermediate metabolites. Further, there is a disruption in tryptophan catabolism that prevents adequate immune cell activation in the liver. The chronic cellular damage cannot be appropriately cleared due to a lack of immune checkpoint activation. This mouse model is an excellent example of how a disruption in small molecule metabolism can significantly impact immune function.

Disruption of hepatic one-carbon metabolism impairs mitochondrial function and enhances macrophage activity in methionine-choline-deficient mice.

One-carbon metabolism is a collection of metabolic cycles that supports methylation and provides one-carbon bound folates for the de novo synthesis of purine and thymidine nucleotides. The methylation of phosphatidylethanolamine to form choline has been extensively studied in the context of fatty liver disease. However, the role of one-carbon metabolism in supporting nucleotide synthesis during liver damage has not been addressed. The objective of this study is to determine how the disruption of one-carbon metabolism influences nucleotide metabolism in the liver after dietary methionine and choline restriction. Mice (n=8) were fed a methionine-choline-deficient or control diet for 3 weeks. We treated mice with the compound alloxazine (0.5 mg/kg), a known adenosine receptor antagonist, every second day during the final week of feeding to probe the function of adenosine signaling during liver damage. We found that concentrations of several hepatic nucleotides were significantly lower in methionine- and choline-deficient mice vs. controls (adenine: 13.9±0.7 vs. 10.1±0.6, guanine: 1.8±0.1 vs. 1.4±0.1, thymidine: 0.0122±0.0027 vs. 0.0059±0.0027 nmol/mg dry tissue). Treatment of alloxazine caused a specific decrease in thymidine nucleotides, decrease in mitochondrial content in the liver and exacerbation of steatohepatitis as shown by the increased hepatic lipid content and altered macrophage morphology. This study demonstrates a role for one-carbon metabolism in supporting de novo nucleotide synthesis and mitochondrial function during liver damage.

One-Carbon Metabolism in Fatty Liver Disease and Fibrosis: One-Carbon to Rule Them All.

Nonalcoholic fatty liver disease (NAFLD) is a term used to characterize a range of disease states that involve the accumulation of fat in the liver but are not associated with excessive alcohol consumption. NAFLD is a prevalent disease that can progress to organ damage like liver cirrhosis and hepatocellular carcinoma. Many animal models have demonstrated that one-carbon metabolism is strongly associated with NAFLD. Phosphatidylcholine is an important phospholipid that affects hepatic lipid homeostasis and de novo synthesis of this phospholipid is associated with NAFLD. However, one-carbon metabolism serves to support all cellular methylation reactions and catabolism of methionine, serine, glycine, choline, betaine, tryptophan, and histidine. Several different pathways within one-carbon metabolism that play important roles in regulating energy metabolism and immune function have received less attention in the study of fatty liver disease and fibrosis. This review examines what we have learned about hepatic lipid metabolism and liver damage from the study of one-carbon metabolism thus far and highlights unexplored opportunities for future research.

Aged garlic extract supplementation modifies inflammation and immunity of adults with obesity: A randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Obesity is a serious global health issue and often results in low-grade systemic inflammation, increasing the risk for several chronic diseases. If obesity-induced inflammation could be reduced, fewer complications and co-morbidities might occur. OBJECTIVE: To investigate whether daily supplementation with aged garlic extract (AGE) could reduce chronic inflammation and improve immune function in adults with obesity. METHODS: Fifty-one healthy adults with obesity (mean age 45.6 ± 1.6 years, mean BMI 36.1 ± 0.9 kg/m2) were recruited to participate in a parallel, double-blind, placebo-controlled, randomized study. After being matched by BMI, participants were randomized into the AGE supplementation or placebo group. Participants were asked to take a divided daily dose of 3.6 g AGE or placebo, with food for 6 weeks. Blood lipid and inflammatory markers were assessed at baseline and after 6 weeks of supplementation. Additionally, peripheral blood mononuclear cells (PBMC) were isolated from whole blood and used to detect changes in immune cell populations and levels of cytokine secretion. A one-way ANCOVA was performed to evaluate differences between the two groups, controlling for respective baseline values. RESULTS: At the end of study, serum IL-6 (p = 0.04) and TNF-α (p = 0.05) of participants consuming AGE were significantly lower than those consuming the placebo capsules. PBMC flow cytometry results showed that participants from the AGE group had a higher proportion of γδ-T cells (p = 0.03) and a lower proportion of NKT cells (p = 0.02) in the total population of lymphocytes. There was no difference in percentage of NK cells between the two groups. A significant difference in blood LDL concentration was also observed (p = 0.05). Total cholesterol and non-HDL cholesterol tended to differ between participants from the AGE group and those from the placebo group, although values did not achieve statistical significance. CONCLUSION: Six weeks of AGE consumption modulated immune cell distribution, prevented the increase of serum TNF-α and IL-6 concentrations and reduced blood LDL concentration in adults with obesity. AGE, taken consistently, may be beneficial in preventing the development of chronic diseases associated with low-grade inflammation in adults with obesity. Registered under ClinicalTrials.gov with the identifier code NCT01959646.